I never used pET20b, but I found on the Internet that it expresses inserts constitutively. Since the cloned protease should be toxic to cells, its constitutive expression might prevent the formation of colonies. But there might be millions of other reasons. Why did you use pET20b?
http://www.biovisualtech.com/bvplasmid/pET-20b%28+%29.htm Alex On Jul 13, 2011, at 5:54 PM, Wonjin Bae wrote: > Hi, all > > Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and > pGEX4T3 vector. > Then, these two construct were transformed to BL21(DE3) expression host. > DNA sequencing results were accurate. > > In case of pGEX, many colony was formed and shown the high level of > expression. > But, colony was not shown in pET20b case. (no one colony) > In case of mock-pET20a vector transformed very well. > > What's the problems? I never experieced transfomation problems. > Does anyone know how to solve the this problems? > > Thanks a lot !! > Genie,
